Advances in molecular biology and genetic engineering led to the development of “Reverse Genetics”, a process of generating recombinant viruses from a cloned complimentary DNA (cDNA) copy of a viral genome. It has helped understand the molecular determinants related to virus attenuation, tissue tropism virulence factors and in recent years, accelerated the development of virus vaccines by enabling easy modification of viral genomes through manipulation of its cDNA. Reverse genetics has made it possible to produce recombinant viruses with attenuating mutations or chimeric viruses expressing heterologous genes for use as new viral vaccines or therapeutic agents.
Morbilli Viruses (Measles Virus and Rinderpest Virus)
MV and RPV are members of the genus morbillivirus of family Paramyxoviridae. Their genetic information is encoded on a single stranded RNA genome of antisense polarity and comprises 15894(MV) and 15882 (RPV) nucleotides respectively. Their genome has unique highly conserved 3 ′ and 5 ′ termini called leader and trailer respectively and encodes 6 genes—N (nucleocapsid protein), P (phosphoprotein), M (matrix protein), F (fusion protein), H (hemagglutinin) and L (large protein=polymerase)—separated by similarly conserved intergenic sequences. In infected cells, viral RNA dependent RNA polymerase (RDRP) initiates transcription of genomic RNA from its promoter present within the leader sequence and produces messenger ribonucleic acid (mRNA) molecules which are translated into corresponding proteins by the cellular ribosomes. At some point in the viral lifecycle and after adequate pools of viral proteins are synthesized, the RDRP enzyme switches mode and initiates replication from another promoter present within the leader sequence of the genomic RNA. Although the exact mechanism(s) regulating the initiation of transcription or replication of viral RNA and transcription start and stop at gene boundaries is poorly understood, the conserved sequences which serve as promoters for transcription and replication and sequences which dictate the transcription start and end at each gene boundary are clearly defined in case of most negative stranded RNA viruses in general and MV and RPV in particular. It is well established that addition of these sequences to any unrelated RNA molecule forms a “virus genome like replicon” which can be transcribed and replicated by its cognate RDRP.
MV causes an acute febrile illness in infants and young children. Its prevalence can be controlled very effectively by vaccination. Most of the currently used live attenuated vaccines including the Schwartz, Moraten, and Edmonston-Zagreb strains are derived from the original Edmonston strain (Enders and Peebles, 1954) by multiple passages in non human cells (Enders, 1962). However, according to the estimates of the World Health Organisation (WHO), one million young children die every year from measles mainly in developing countries. But in recent years developed countries such as the USA with incomplete adherence to vaccination have seen emergence of measles related deaths (Clements and Cutts, 1995). For a recent discussion of MV vaccinology including future trends see Norrby (1995). During the past 60 odd years, Measles vaccine has been administered in over 700 million children and has proved to be highly effective, usually providing life-long immunity against MV reinfection.
RPV causes cattle plague—an infectious viral disease of cattle, buffalo and other wild life species and is mainly India, Africa and other tropical countries. It is characterized by fever, oral erosions, diarrhea, lymphoid necrosis and high mortality. Two vaccines—Plowright (Plowright and Ferris, 1962) and Lapinized (Scot 1963) have been widely used to protect against rinderpest. The Plowright vaccine derived by attenuation of RBOK strain of RPV has proved to be most effective (Baron et al, 2005). Wide spread use of these vaccines helped irradicate RPV from several countries including India by 2000. However, its reemergence in 2003 has led to resumption of mass vaccinations of cattle and other susceptible animals (Kock et al, 2006).
The proven safety and efficacy of these vaccines, supports their use as an ideal vector for the expression of heterologous genes. Reverse genetics offers a powerful approach for developing recombinant MV or RPV useful as potential vaccines against unrelated diseases and/or therapeutic agents in man and animals.
Reverse genetics was first used to generate RNA viruses by Racaniello and Baltimore in 1981 in case of Poliovirus. Subsequently, several other positive-sense RNA viruses were generated using synthetic RNA produced by T7 or T3 RNA polymerase (Racaniello, V. R. & Baltimore, D., 1981) from a cloned cDNA. Generation of negative stranded RNA viruses however, proved more difficult. Unlike positive stranded RNA viruses, the genome of negative sense viruses cannot be translated by host cells and is not infectious. It must be supplied in the form of ribonucleoprotein (RNP) complexes containing the nucleoprotein and the viral RDRP proteins to allow its transcription and replication and subsequent virus formation. Enami et al, (1990) developed the first reverse genetics system to produce influenza virus (which consists of 9 genomic RNA subunits). Its RNPs are small in size and can be assembled in vitro from RNA and required viral proteins—N and the polymerase components. Initially an artificial RNA carrying a reporter gene—chloramphenicol acetyl transferase (CAT) sequence embedded in viral non-coding terminal sequences of the influenza virus genome subunit was used (Luytjes et al., 1989). Later, single authentic or altered genome subunit RNAs transcribed in vitro from cloned DNA were also used (Enami and Palese, 1991). The assembled RNPs replicated and transcribed upon transfection into influenza-infected cells, as monitored by CAT production and by rescue of a influenza virus, respectively. Purification of virus containing the introduced subunit from the vast excess of non-reassorted virus in some cases can be accomplished by selection, for example, using a specific neutralising antibody directed against the protein encoded by the cognate subunit of the helper virus.
The RNP s of nonsegmented negative-strand RNA viruses (Mononegavirales) contains in addition to N protein, the assembly and polymerase cofactor phosphoprotein (P) and the viral RNA polymerase (large protein L) and are more difficult to assemble in vitro from synthetic RNA and individual proteins. Therefore, many researchers preferred to use smaller subgenomic RNAs (viral minigenomes) containing the essential sequences of viral genome produced during virus lifecycle were used. They were then substituted by artificially transcribed RNA molecules from DNA constructs containing reporter genes and viral essential non-coding sequences (replicons). Replication of such replicons carrying the CAT coding sequence and viral noncoding terminal sequences was achieved for Sendai virus (Park et al., 1991), Sendai. virus (SeV), respiratory syncytial virus (Collins et al., 1993; Collins et al., 1991), human parainfluenza virus 3 (Dimock and Collins, 1993), rabies virus (RV) (Conzelmann and Schnell, 1994) and MV (Sidhu et al., 1995).
A similar system was used to rescue vesicular stomatitis virus (VSV) (Lawson et al., 1995; Schnell, et al, 1994) and rabies virus (RV) entirely from a full length cDNA clone of viral genome under the control T7 RNA polymerase promoter. The components of the viral polymerase complex including the nucleoprotein (NP) were provided from protein expression plasmids that were controlled by T7 RNA polymerase promoter. Soon other researchers also reported generation of non-segmented negative-sense RNA viruses from cloned genomic cDNA for vesicular stomatitis virus (Whelan, et al, 1995), measles virus (Radecke et al, 1995), respiratory syncytial virus (Collins, et al, 1995), sendai virus (Garcin, et al, 1995; Kato et al, 1996), rinderpest virus (Baron & Barrett 1997), human parainfluenza virus (Hoffman et al, 1997; Durbin et al, 1997), simian virus (He et al, 1997), newcastle disease virus (Peeters, et al, 1999) and human severe acute respiratory syndrome corona virus (Yount, et al, 2003).
These demonstrations and other studies of reconstitution of RNA dependent RNA polymerase (RDRP) enzyme activity and its ability to rescue corresponding RNA viruses or non-viral reporter proteins from minireplicons have establish the RDRP enzyme as a powerful versatile system for expression of recombinant proteins either alone or as integral parts of rescued viruses.
The most common methodology used for this purpose, uses transfection of multiple plasmids—one expressing the substrate RNA (a cDNA encoding viral genome or an artificial replicon) and others expressing the viral RDRP complex proteins—viz. the nucleocapsid (N or NP protein), the phosphoprotein (P) and the large polymerase (L) protein and an external T7 RNA polymerase (T7RNAP) to allow expression from these plasmids. The T7 RNAP is used for multiple reasons—(1) its high efficiency, (2) its ability to synthesize RNA with correct 5′ terminus identical to viral genome and (3) its ability to transcribe DNA molecules within the cytoplasm thus eliminating modifications of vRNA by RNA splicing, polyadenylation or other mechanisms.
T7RNAP is not a mammalian enzyme. Therefore Pattnaik et al, (1990) used a recombinant attenuated vaccinia virus (VV) (e.g. MVA/T7). It was used for recovery of VSV (Lawson et al, 1995) and rabies virus (Conzelman, U.S. Pat. No. 6,033,886), RSV (Collins et al, 1995); the SV5 (He et al, 1997), HPIV-3 (Durbin et al, 1997), rinderpest virus (Barn and Barrett 1997) and measles virus (Schneider et al, 1997), mumps virus (Clarke et al, 2000), CDV (Gassen et al, 2000), HPIV-2 (Kawano et al, 2001) and BPIV-3 (Schmidt et al, 2000). Similarly, a recombinant fowlpox virus expressing T7RNAP has also been used to supply T7RNAP for recovery of newcastle virus (NDV) (Peeters et at. 1999) and of a chimeric rinderpest virus (Das et al. 2000).
The recombinant viruses produced using this approach are mixed with vaccinia virus and are difficult to purify which can be a major problem—especially if the recombinant viruses are required for preparing immunogenic compositions or gene therapy vectors. Moreover, this helper vaccinia virus kills the host cells limiting the efficiency of recombinant virus production. Therefore, it would be desirable to eliminate the use of helper virus supplying T7 RNA polymerase. Three different approaches have been used to eliminate the use of externally supplied T7RNAP altogether.
Radecke et al, (1995) produced a helper cell line constitutively expressing T7RNAP and Measles virus (MV) N and P proteins (WO 97/06270) and introduction of a plasmid encoding the entire (+) strand sequence of MV genome linked to T7RNAP promoter and another plasmid encoding MV L protein alone is sufficient to rescue recombinant MV. However, the efficiency of this helper cell line is usually limited and requires to be enhanced by giving a heat shock (Parks et al, 1999). Also, this cell line is only useful for rescue of MV. In contrast, the helper BHK-21 cell line (BSR 17/5) stably expresses only the T7RNAP and can be used for rescue of different viruses as shown in case of BRSV (Buchholz et al. 2000), rabies virus (Finke and Conzelmann 1999), VSV (Harty et al. 2001), NDV (Romer-Oberdorfer et al. 1999), and Ebola virus (Volchkov et al. 2001). It can be used to reconstitute RDRP of any virus by co-transfecting with plasmids encoding appropriate N, P and L proteins.
Second approach involves the use of RNA polymerase I (RNAPI). RNAPI is usually involved in transcription of ribosomal genes in mammalian cells. The RNAs synthesized by RNAPI do not contain the 5′ methyl cap structure and 3′ poly-A tail. The transcription initiation and termination signals for RNAPI are precisely defined and RNA molecules produced by inserting viral genomic or genome like cDNA molecules in between rRNA promoter and terminator signals possess authentic viral 5′ and 3′ ends, does not require further processing and can be used as a substrate directly by viral RDRP if expressed. (Zobel et al, 1993, Nucleic acids research, 21:3607-3612; Flick and Petterson, 2001, J. Virol. 75: 1643-1655;). Therefore, RNAPI transcription has been used to synthesize viral genomic or genomic like cDNA from plasmids and used for rescue of viruses in case of Influenza virus (Neumann et al, 1999), Borna disease virus and MV (Martin et al, 2006,1 Viral. 80:5708-5715).
More recently, Martin et al, (2006) have used a third strategy to express viral genomic RNA from transcripts produced by RNA polymerase II (RNAP II). They placed a hammerhead ribozyme immediately upstream of and a genomic hepatitis delta virus ribozyme immediately downstream of the virus genomic sequence. These ribozymes cleaved a genomic RNA with authentic 3′ and 5′ ends from the RNA transcribed by RNAP II.
Such strategies eliminate the need for helper virus but still require separate helper plasmids expressing the viral N, P and L proteins. Transfection of so many plasmids simultaneously in a cell and ensuring useful levels of expression of the desired proteins for efficient reconstitution of RDRP can be difficult. Availability of a single helper plasmid expressing all desired genes will help increase the efficiency of virus rescue by ensuring that all transfected cells will receive the entire complement of helper proteins necessary for reconstitution of RDRP enzyme activity.
This requirement for multiple plasmids has also restricted the use of RDRP based systems to virus rescue, where as studies with artificial replicons encoding reporter proteins has shown that RDRP mediated expression systems can allow high levels expression of recombinant proteins. Availability of a single helper plasmid/reagent to supply the required N, P and L proteins will help expand the scope of using RDRP enzyme for large scale expression of recombinant proteins. Therefore, there exists a need in the art for new simpler methods and reagents which will allow efficient reconstitution of RDRP activity and its exploitation for expression of recombinant proteins, RNA molecules and/or rescue of recombinant viruses.
Here, we describe the preparation and use of simple easily manipulatable plasmid vector systems which can be used for reconstitution of RDRP enzyme activity and its rescue for expression of recombinant proteins, RNA molecules and rescue of recombinant viruses. For this purpose, we have used the RDRP system of 2 viruses—Measles virus (MV) and Rinderpest virus (RPV) as models. These plasmids can be easily modified to express either non-viral proteins, RNA molecules or the entire viral genomes. This vector system will be useful in development of applications related to protein expression and/or generation of recombinant modified viruses (virus rescue) expressing additional proteins and/or RNA molecules useful for vaccination or other therapeutic purposes.